Nucleic acid polymers such as DNA and RNA are essential to the transmission of genetic information from one generation to the next and in the routine functioning of all living organisms. Accordingly these molecules are the objects of intense study and a number of techniques have been developed to study of these molecules. These methods include but are not limited to methods for identifying the presence of a specific polynucleotide sequence in a given sample and methods designed to measure the number of specific nucleic acid molecules originally present in a given sample.
Practical uses for these techniques include identifying specific species and relationships between various species based upon similarities in oligonucleotide sequences. Other uses include diagnosing disease by identifying specific sequences in a given sample as indicative of a given pathology. Still other uses, too numerous to mention, include identifying individuals with a predisposition for developing a specific pathology as well as assessing the efficacy of proposed treatment regimes based on the presence of specific polynucleotides in a given patient's genome.
One of the most widely used and powerful techniques for the study and manipulation of oligonucleotides is the polymerase chain reaction (PCR). PCR is a primer extension reaction that provides a method for amplifying specific nucleic acids in vitro. This technique was first described in 1987. PCR can produce million fold copies of a DNA template in a single enzymatic reaction mixture within a matter of hours, enabling researchers to determine the size and sequence of target DNA. This DNA amplification technique has been widely used for cloning and other molecular biological manipulations. Further discussion of PCR is provided in Mullis et al., Methods Enzymol. (1987); and Saiki et al., Science (1985).
One PCR based technique that is particularly useful is Quantitative PCR (qPCR). Briefly, the mechanism of qPCR is based on the fact that PCR amplifies a target DNA in an exponential manner. By running a PCR reaction and measuring the total number of DNA copies at given points during the course of the amplification reaction, one can retroactively calculate the amount of starting DNA material.
Various methods have been developed for determining the amount of PCR product made without having to stop the PCR run or even to sample the reaction during a given PCR run. One such method follows the course of the PCR run in real time by measuring the amount of product at each cycle of DNA synthesis. This process is referred to as real-time PCR. Because of its great sensitivity and because measurements can be made with the sample still in the PCR thermocylcer, various fluorescence-based assays that monitor the formation of PCR products have been developed.
In real-time PCR, a fluorogenic molecule is used to monitor the progress of target DNA amplification. Depending on the mode of signal generation, the fluorogenic molecule may either be a fluorogenically labeled oligonucleotide or a simple fluorogenic DNA-binding dye. Real-time qPCR using a DNA-binding dye is primarily used for research applications while that using an oligonucleotide-based probe is used in diagnostics or applications where highly specific nucleic acid detection is essential. The most widely used fluorogenic probes are commercially called TaqMan® probes, which are oligonucleotides containing a covalently labeled fluorescent reporter dye and a fluorescence quencher molecule. By having its sequence complementary to a target sequence, a TaqMan® robe is capable of binding to a region of the target sequence via hybridization. Before hybridization, a TaqMan® probe assumes a random conformation, in which the fluorescence of the reporter dye is quenched by the fluorescence quencher via fluorescence resonance energy transfer (FRET) due to the proximity of the reporter and quencher molecules. FRET is dependent on the inverse sixth power of the distance between the reporter and quencher. Consequently, the level of fluorescence exhibited by a TaqMan® probe is highly sensitive to the distance between the quencher and the reporter. On hybridization to a target sequence, the reporter dye and the quencher are separated by a longer distance, resulting man increase of fluorescence due to less efficient FRET. The degree of fluorescence increase is proportional to the amount of target DNA present, thereby making it possible to monitor the amount of DNA in real-time as the PCR proceeds. When a PCR is carried out using a DNA polymerase possessing a 5′-exonuclease domain, the hybridized TaqMan® probe will be cleaved by the exonuclease, resulting in the permanent separation of the reporter dye and quencher. Because the reporter dye and quencher are completely separated, the fluorescence of the reporter dye is fully released. For this reason, real-time qPCR relying on the hydrolytic cleavage of a TaqMan® probe is generally more sensitive than that relying on the hybridization of the probe. Since a TaqMan® probe can both monitor the progress of the PCR and verify the identity of the amplified target at the same time, TaqMan®-based real-time qPCR makes a post PCR verification step unnecessary, providing the high specificity required for demanding applications such as medical diagnosis and prognosis. However, TaqMan® probes are usually expensive and time-consuming to synthesize. Furthermore, due to their complex design and the uniqueness of each probe, the quality of TaqMan®-like probes could vary considerably from lab to lab and from batch to batch, potentially leading to inconsistent PCR results from time to time.
Given the importance of oligonucleotide-based probes, there remains a considerable need for further improved methods for the designs and manufacturing of these probes. One object of the present invention is to provide alternative oligonucleotide-based probes and efficient methods for making and using the same.